Multi-omic profiling reveals the endogenous and neoplastic responses to immunotherapies in cutaneous T cell lymphoma

Summary Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA+CD39+ T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA+CD39+ T cells in non-responders and activated cytotoxic CLA+CD39+ T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials.

FDR-adjusted Wilcoxon signed rank.T) % neoplastic T cells of total PBMCs by timepoint for subjects with late timepoint data colored by therapeutic response.Crossbar indicates mean.U) Probably of generation of TCR sequence for neoplastic TCRs (dots) and total normal repertoires (violin).

Figure S1 :
Figure S1: Quality control, clustering, and disease status differencesrelated to Figure 1 A) Subject age by disease status.Crossbar indicates mean.Kruskal-Wallis.B) Subject age by response.Crossbar indicates mean.Wilcoxon rank sum.C) Subject age by response and disease status.Crossbar indicates mean.FDR-corrected Wilcoxon rank sum.D) Subject gender by disease status.Fisher's exact.E) Subject gender by response.Fisher's exact.F) Subject gender by response and disease status.FDR-corrected Fisher's exact.G) Exemplative batch correction of three protein channels.Violins represent cell distributions from a common control sample run with each batch.H) Median expression of proteins by cell types from an equal sampling of subjects at baseline.Canonical markers highlighted in red.I) UMAPs of individual CTCL subjects in this study (top row) or a public scRNAseq dataset (middle, bottom rows) colored by cell type (top, middle rows) or by the detection of the neoplastic TCR clone (bottom row).J) % neoplastic T cells of PBMCs or of total T cells by clinical flow cytometry, mass cytometry, and TCR sequencing.Fitting lines calculated by linear regression.R-and p-values calculated by Pearson method.K) Evenness and richness scores of BL TCR repertoires by status with or without neoplastic T cell clones.Wilcoxon rank sum.L) BL TCR counts as calculated by Adaptive Biotechnology template counting by disease status.Crossbar indicates mean.Wilcoxon rank sum.M) Median expression of proteins by non-neoplastic T cell clusters, from an equal sampling of subjects at BL. N) Percent of T cell clusters out of non-neoplastic T cells at baseline, separated by disease status.Crossbar indicates mean.FDR-corrected Kruskal-Wallis.

Figure S3 :
Figure S3: In vivo effects of interferon-gammarelated to Figure 3 A) %PD-L1 + cells by cell type and timepoint.Crossbar indicates mean.Differences across timepoints were not significant (q>0.1).FDR-corrected Wilcoxon signed rank.B) Log 2 expression of serum proteins and tumor mRNA by timepoint and colored by response.Crossbar indicates mean.Differences across timepoints were not significant (q>0.1).FDR-corrected Wilcoxon signed rank.C) Log2 expression of serum proteinsand tumor mRNA at C1 by therapeutic response.Crossbar indicates mean.FDR-corrected Wilcoxon rank sum.D) Mean log2 change in expression of IFNg response genes in tumor mRNA between BL and C1, by subject, colored by response.E) Log2 change in expression of IFNg response genes in serum proteins between BL and C1, by subject.F) Log2 expression of serum protein and tumor mRNA by timepoint.Crossbar indicates mean.Wilcoxon signed rank.G) Log2 change in expression of serum proteins between BL and C1, by subject.All proteins were significantly differentially expressed (p<0.05 & |effect size|>1).Wilcoxon signed rank.H) Log2 expression of tumor mRNA by timepoint.Crossbar indicates mean.Wilcoxon signed rank; |effect size|>1.I) Difference in cell type abundance between BL and C1.Differences across timepoints were not significant (q>0.1).FDR-corrected Wilcoxon signed rank.J) %HLA-DR + of CD14 + monocytes at BL by cohort and disease status.Crossbar indicates mean.FDRcorrected Wilcoxon rank sum.K) %HLA-DR + of CD14 + monocytes at C1 by cohort and disease status.Crossbar indicates mean.Wilcoxon rank sum.L) %CXCR3 + of T cell populations, separated by timepoint.* indicates q<0.1.FDR-corrected Wilcoxon signed rank.Boxplots represent interquartile range (IQR); horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.M) % T cell populations of non-neoplastic T cells by timepoint.Crossbar indicates mean.FDR-corrected Wilcoxon signed rank.N) % Ki-67 + of T cell populations by timepoint.Crossbar indicates mean.FDR-corrected Wilcoxon signed rank.O) Mean expression of metabolic markers by CD8 + EMRA T cells across timepoints.Both markers weresignificantly differentially expressed between timepoints (q<0.1).FDR-corrected Wilcoxon signed rank.P) TCR clones, colored by indicated criteria, organized into metaclones.Q) % of TCRs by clone, colored by subject, separated by metaclone.R) V and J gene usage and CDR3 sequence motifs of the metaclones indicated in (Q).The top sequence logo shows the information relative to amino acids found in a background set of randomly sampled CDR3 sequences with the same V and J genes.

Figure S4 :
Figure S4: In vivo effects of anti-PD-1 -related to Figure 4A) %PD-1 + T cell populations, separated by timepoint.* indicates q<0.1.FDR-corrected Wilcoxon signed rank.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.B) Log2 expression of serum proteins and tumor mRNA by timepoint.Crossbar indicates mean.Differences across timepoints were not significant (q>0.1).FDR-corrected Wilcoxon signed rank.C) Pearson correlation between proteins expressed on non-neoplastic T cells at C2, post-anti-PD-1 treatment.D) Exemplative staining of T cells from an equal sampling of non-neoplastic T cells pre-and post-anti-PD-1 treatment.E) Composition of T cell subsets comprising total CD8 + T cells and CMV-specific CD8 + T cells in two subjects.F) Exemplative staining of T cells from an equal sampling of non-neoplastic T cells pre-and post-anti-PD-1 treatment.G) % + activation markers across T cell populations by timepoint.FDR-corrected Wilcoxon signed rank.H) The number of significantly differentially abundant clones (q<0.1) between indicated timepoints as calculated by FDR-corrected Fisher's exact test.P-value calculated from Wilcoxon signed rank test.I) One minus the Horn-Morista index of the non-neoplastic TCR repertoire similarity of CTCL subjects at indicated timepoints by response.Crossbar indicates mean.FDR-corrected Wilcoxon rank sum.J) Effect size difference for genes not significantly differentially expressed after multiple hypothesis correction (p<0.05;q>0.1) between BL and C2.Uncorrected/FDR-corrected Wilcoxon signed rank.K) Log2 expression of serum proteins significantly differentially expressed (q<0.1) between BL and C2.Crossbar indicates mean.FDR-corrected Wilcoxon signed rank.

Figure S5 :
Figure S5: Identification of immune features stratifying responders and non-respondersrelated to Figure 5 A) % variance explained by PC1 within each immune module by timepoint.Dots indicate median.B) Out-of-bag errors across different seeds of the random forest models generated to predict therapeutic response using immune modules by timepoint.Crossbar indicates mean.Lower error indicates improved accuracy.C) % Tregs of T cells and %Ki-67 + Tregs at BL by response.Crossbar indicates mean.FDR-corrected Wilcoxon rank sum.D) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying therapeutic response at C2. Uncorrected Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in responders and features enriched in non-responders.E) Median of mean expression of genes identified in (D) by cell population.F) Gini index of immune module predictors separating response and non-response at the BL timepoint.Dashed red line indicates threshold set for immune modules shown in (G).Colors correspond to immune modules in (G).G) Effect size of underlying features comprising immune modules selected in (F).Bar color denotes immune module membership.Features are ordered by Gini index.H) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying therapeutic response at BL. Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in responders and features enriched in non-responders.I) % + of activation markers by CD8 + CLA + EM T cells at BL by response.Crossbar indicates mean.No comparison was statistically significant (q>0.1).FDR-corrected Wilcoxon rank sum test.J) Gini index of immune module predictors separating response and non-response in the C2-BL timepoint.Dashed red line indicates threshold set for immune modules shown in K. Colors correspond to immune modules in (K).K) Effect size of underlying features comprising immune modules selected in (J).Bar color denotes immune module membership.Features are ordered by Gini index.L) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying therapeutic response at C2-BL difference.Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in responders and features enriched in non-responders.

Figure S6 :
Figure S6: Mono and combination therapy induce differential abundance of PD-1 -related to Figure 5 A) Out-of-bag errors across different seeds of the random forest models generated to predict therapy regimen using immune modules at the C2-BL difference.Crossbar indicates mean.Lower error indicates improved accuracy.B) Gini index of immune module predictors separating mono and combination therapy in the C2-BL timepoint.Dashed red line indicates threshold set for immune modules shown in (C).Colors correspond to immune modules in (C).C) Effect size of underlying features comprising immune modules selected in (B).Bar color denotes immune module membership.Features are ordered by Gini index.D) Log2 expression of serum PD-1 by clinical trial and timepoint.Crossbar indicates mean.FDR-corrected Wilcoxon signed rank.E) Log2 expression of tumor pdcd1 mRNA by clinical trial and timepoint.Crossbar indicates mean.FDRcorrected Wilcoxon signed rank.F) %PD-1 + of total T cells by clinical trial and timepoint.Crossbar indicates mean.FDR-corrected Wilcoxon signed rank.G) Difference in %PD-1 + (top row) or %HLA-DR + (bottom row) between C2 and BL in T cell populations by clinical trial.Crossbar indicates mean.FDR-corrected Wilcoxon rank sum.H) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying response to mono and combination therapy at C2-BL difference.Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in monotherapy and features enriched in combination therapy.

Figure S7 :
Figure S7: Identification of immune features stratifying MF and SSrelated to Figure 6 A) Out-of-bag errors across different seeds of the random forest models generated to predict disease status using immune modules, separated by timepoint.Crossbar indicates mean.Lower error indicates improved accuracy.B) Gini index of immune module predictors separating MF and SS at the BL timepoint.Dashed red line indicates threshold set for immune modules shown in Fig. 6A.Colors correspond to immune modules in Fig. 6A.C) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying MF and SS subjects at BL. Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in MF and features enriched in SS.D) % macrophages of immune cells in tumor samples at BL. Crossbar indicates mean.Wilcoxon rank sum.E) % immune cells of total cells in tumor samples at BL. Crossbar indicates mean.Wilcoxon rank sum.F) %PD-1 + of indicated T cell populations in tumor samples at BL. Crossbar indicates mean.FDR-corrected Wilcoxon rank sum.G) %HLA-DR + of indicated tumor-present T cell populations at BL by subject.H) CXCL13 serum protein level and abundance of indicated cell populations at BL. I) Expression of pS6 for indicated T cell populations drawn from an equal sampling of cells from HC, MF, and SS subjects at BL. J) Gini index of immune module predictors separating MF and SS at the BL timepoint.Dashed red line indicates threshold set for immune modules shown in (K).Colors correspond to immune modules in (K).K) Effect size of underlying features comprising immune modules selected by model differentiating MF and SS subjects at C2. Bar color denotes immune module membership as in (J).Features are ordered by Gini index.L) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying MF and SS at C2. Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in MF and features enriched in SS.M) Gini index of immune module predictors separating MF and SS at C2-BL difference.Dashed red line indicates threshold set for immune modules shown in (N).Colors correspond to immune modules in (N).N) Effect size of underlying features comprising immune modules selected by model differentiating MF and SS subjects at C2-BL difference.Bar color denotes immune module membership as in (M).Features are ordered by Gini index.O) Z-scores of significantly differentially expressed features (p<0.05& |effect|>1) stratifying MF and SS subjects at C2-BL difference.Wilcoxon rank sum.Boxplots represent IQR; horizontal line indicates median; whiskers extend to the farthest data point within 1.5 × IQR.Dashed line separates features enriched in MF and features enriched in SS.